Elucidation of the structure and function of proteins involved in the regulation of RNA synthesis in animal cells is necessary for understanding cell differentiation and development. Experiments detailed in this proposal address fundamental questions about the structure, function, and evolution of Xenopus transcription factor IIIA (TFIIIA). TFIIIA binds specifically to 5 S ribosomal RNA genes in the South African clawed toad, Xenopus laevis, and regulates the transcription of these genes by RNA polymerase III. TFIIIA contains zinc and requires the metal for specific binding to the 5 S RNA gene. The amino acid sequences of TFIIIA and other eukaryotic transcription factors contain potential zinc binding sites of similar structure. Postulated zinc binding sites encoded in TFIIIA cDNA will be subjected to in vitro mutagenesis to determine their necessity in TFIIIA function. Mutant proteins will be expressed from these cDNAs in E. coli and the isolated proteins will be assayed for their ability to protect the 5 S RNA gene from DNase I digestion. In vitro mutagenesis will also be used to determine whether loop structures in TFIIIA are required for DNA binding, to identify amino acids involved in protein-DNA contacts, and to identify structures in the C-terminal region of TFIIIA involved in the promotion of transcription. TFIIIA-like proteins from other species will be isolated and characterized structurally and functionally. The genes for these TFIIIA-like proteins will be isolated and sequenced to determine the degree of conservation in their amino acid sequences and intron-exon structures.